No. PO-88

・日時：2019年7月22日(月)

・Poster Session VIII "Cancer ３"

17:20-17:35

・会場：安田講堂 Poster会場

・３題 (各5分発表： 3分口演+2分討論）

Design of chimeric antigen receptors affects the characters of CAR-T cells Yasunori Amaishi*1, Sachiko Okamoto*1, Tatsuji Enoki*1, Hiroshi Shiku*2, Junichi Mineno*1

*1 Project Development Department, Takara Bio Inc.

*2 Department of Immuno-Gene Therapy,

Mie University Graduate School of Medicine

Non-specific activation of CAR-T cells

CAR-T cells expressed the activation marker CD25 without antigen stimulation, and CD25 expression

levels were correlated with CAR expression levels regardless of the intracellular domain of CARs.

Correlation between CD25-expression and CAR-expression

Introduction

CAR-T cell therapy using chimeric antigen receptor (CAR) transduced T-cells, has recently attracted much atten-

tion as one of effective cancer immunotherapies. In particular, CAR-T cell therapy targeting the CD19 antigen of

B-cell tumor has been shown to achieve very high response rate to hematologic cancer, and CAR-T targeting

CD19 have begun to be approved as new drugs. However, the clinical effect of CAR-T on solid tumors is limited,

and a high recurrence rate is pointed out for blood tumors, there is still a need to produce effective CAR-T cells in

the body for a long duration while retaining a high therapeutic effect.

We have been developing CAR constructs focusing on antigen non-specific activation of CAR-T cells due to the

design of CARs. Since CAR is an artificial protein in which the single-chain antibody (scFv) and the signal domains

are directly linked, CAR-T cells have antigen non-specific activation caused by interaction between CAR and other

cellular molecules.

Here, in order to analyze the influence of the extracellular design of CARs on T cells in more detail, we construct-

ed several CARs having different design (the order of scFv variable regions, the linker sequence between the vari-

able regions, the type and length of extracellular spacer region and the transmembrane domains, etc.) and evalu-

ated their characteristics in T cells.

CAR-T cell therapy

Influence of CAR design on the characters of T-cells

Comparison of ScFv design

The nonspecific activation and the property of CAR-

T cells were also differd depending on the order of

V region and the linker sequence in scFv, although

the effect was not as great as that of the hinge.

Comparison of Hinge region 1

Comparison of Hinge region 2

Hinge region was crucial for CAR expression on T cells.

The strength of non-specific activation differed depending on the hinge type, highly activated

CAR-T cells showed higher expression of exhaustion markers, reduction of naive phenotype,

reduction of cytokine production ability, and reduction of cytotoxic activity.

Summary

・ CAR-T cells showed antigen non-specific activation which was not detected on TCR-T cells.

・ Non-specific activation originated from extracellular design of CARs.

・ The non-specific activation affected the properties of CAR-T cells.

・ Low naïve memory subsets and constant expression of exhaustion markers.

・ Cytotoxicity and cytokine production capacity against antigen expressing cells.

⇒ Need to choose the appropriate CAR design for the effective CAR-T therapy.